Diabetes alters the protein secretome of human adipose‐derived stem cells and promotes tumorigenesis in hepatic cancer cells

MATERIALS AND METHODS Study selection and sample processing hASCs were obtained from the SAT of ageand sex-matched donors undergoing non-acute surgical interventions, such as hernia or cholecystectomy, in a scheduled routine surgery (n=6 per group) using standard protocols.1–4 Briefly, SAT was washed extensively with phosphate buffered saline (PBS) and treated with 0.1% collagenase in PBS+1% bovine serum albumin (BSA) for 1 hour at 37oC with agitation. Digested samples were centrifuged to separate adipocytes from stromal cells. The cell pellet containing the stromal fraction was resuspended in stromal culture medium. The medium was replaced 24 hours after seeding to remove non-adherent cells. When cultures reached approximately 80% confluency at passage 0 (P0), the cells were harvested by trypsin and seeded for the following passage or centrifuged to resuspend the pellet at a concentration of 106 hASCs/ml in cryomedium (10% DMSO, 10% DMEM/F12, 80% bovine calf serum). The cells were dispensed in 1-ml aliquots in cryovials, maintained overnight in a -80oC freezer and then stored in liquid nitrogen until they were required for the individual experiments. All participants gave their informed consent and the study was reviewed and approved by the ethics and research committee of University Hospital Joan XXIII, Tarragona, Spain. Clinical and laboratory data of the participants are shown in Table S1.

the stromal fraction was resuspended in stromal culture medium. The medium was replaced 24 hours after seeding to remove non-adherent cells. When cultures reached approximately 80% confluency at passage 0 (P0), the cells were harvested by trypsin and seeded for the following passage or centrifuged to resuspend the pellet at a concentration of 10 6 hASCs/ml in cryomedium (10% DMSO, 10% DMEM/F-12, 80% bovine calf serum). The cells were dispensed in 1-ml aliquots in cryovials, maintained overnight in a -80ºC freezer and then stored in liquid nitrogen until they were required for the individual experiments. All participants gave their informed consent and the study was reviewed and approved by the ethics and research committee of University Hospital Joan XXIII, Tarragona, Spain. Clinical and laboratory data of the participants are shown in Table S1.
Adipose derived-stem cell culture and conditioned media collection Individual vials of cells were removed from liquid nitrogen, placed in a 37ºC water bath and agitated rapidly but gently until the ice crystals were thawed. Cells were transferred to a 15-ml conical tube containing 5 ml of stromal medium consisting of DMEM/F12, 10% FBS, and 1% antibiotics (penicillin and streptomycin). The cell suspension was centrifuged at 1,200 rpm at room temperature, the supernatant aspirated to remove residual DMSO, and the cell pellet resuspended in stromal medium in preparation for plating. Cell suspensions (P1) were seeded into T-75 cm 2 flasks. Cultures were replenished with fresh stromal medium every 2-3 days as described, 4 and cells were used for experiments from P3. Flow cytometry analysis of cell marker expression revealed that hASCs showed minimal functional and quantitative criteria as established by the International Society of Cell Therapy and the International Federation for Adipose Therapeutics and Science (Positive for CD73, CD90, CD105, and negative for CD14, CD31, CD34, CD45).
The used CM was collected from hASC cultures at P6 as follows: cells were plated at a density of 12,000 cells/cm 2 in 35-mm culture plates and allowed to grow for 24 hours. After this, the plates were first washed with PBS without calcium or magnesium, and then stromal medium without FBS was added to the cells. After 9 h, the CM was collected and frozen at -80ºC until required. Chromatographic gradients run were: 97% buffer A, 3% buffer B to 65% buffer A, 35% buffer B in 120 min at a flow rate of 250 nl/min (buffer A: 0.1% formic acid in water and buffer B: 0.1% formic acid in acetonitrile). The instrument was operated in DDA mode and full MS scans with 1 micro scans at resolution of 60,000 were used over a mass range of m/z 350-2,000 with detection in the Orbitrap.
Following each survey scan the top twenty most intense ions with multiple charged ions above a threshold ion count of 5000 were selected for fragmentation at normalized collision energy of 35%.
Fragment ion spectra produced via collision-induced dissociation (CID) were acquired in the linear ion trap. Digested bovine serum albumin (cat. #P8108S, New England Biolabs) was analyzed between each sample to avoid sample carryover and to assure stability of the instrument, and Qcloud 5 was used to control instrument longitudinal performance during the project. All data were acquired with Xcalibur software v2.2. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository 6 with the dataset identifier PXD029829.
Secretome data analysis Acquired spectra were analyzed using the Proteome Discoverer software suite (v1.4, Thermo Fisher Scientific) and Mascot search engine v2.5 (Matrix Science). 7 Data were searched against the Swiss-Prot human database (release date April 2018) plus a list of common contaminants (refined from MaxQuant contaminants.fasta) and the corresponding decoy entries. For peptide identification, a precursor ion mass tolerance of 7 ppm at the MS1 level was used, trypsin was chosen as enzyme, and up to three missed cleavages was allowed. The fragment ion mass tolerance was set to 0.5 Da for MS2 spectra.
Oxidation of methionine and N-terminal protein acetylation were defined as variable modifications, whereas carbamidomethylation on cysteines was set as a fixed modification. FDR for peptide identification was set to a maximum of 5%.
Protein abundance was estimated using the average area under the chromatographic peak for the three most intense peptides. Differential enrichment was done using the DEP package, which provides an integrated analysis workflow for robust and reproducible analysis of MS proteomics data. 8 Briefly, identified proteins were filtered to keep those detected in all or all but one replicate of at least one condition, resulting in 231 proteins retained. The filtered data were then background corrected and normalized by variance stabilizing transformation. 9 Protein-wise linear models combined with empirical Bayes statistics were used for the differential enrichment analysis using the limma package. 10 In silico protein functional analysis Computational prediction of secreted proteins was performed using SignalP (http://www.cbs.dtu.dk/services/SignalP/) and by UniProt including proteins with the keywords "Signal," "Secreted," or "Extracellular space." If a protein belonged to at least one of these four criteria, it was considered "predicted to be secreted" otherwise "not predicted to be secreted". Known interactions between ligands and receptors were retrieved from MetaCore. Plasma membrane receptors were defined as those having either the UniProt keyword "cell membrane" or Panther protein class "receptor" or GO.CC "plasma membrane". Self-interactions were discarded. 11

Statistical analysis
In vitro experiments were performed 5-6 times and pooled for statistical analysis. Data are presented as mean ± S.E.M. and represent the number of biologically-independent samples. Differences between groups were determined using unpaired Student's t-test to compare two groups (two-tailed, 95% confidence interval). The analyses were performed using GraphPad Prism 8.0.2 software (GraphPad Software Inc.). A p-value < 0.05 was considered statistically significant. For clinical and anthropometrical variables, normally distributed data were expressed as mean ± S.D.